We are studying the mechanism of control of expression of genes of D- galactose transport and metabolism in Escherichia coli. We have previously demonstrated that the members of the gal regulon are negatively regulated to different extents by Gal repressor (GalR) and isorepressor (GalS). We have shown that the promoters, P1 and P2, of the gal operon, are completely repressed if a DNA loop covering the promoter segment is formed by the association of GalR bound to two operators, OE and OI. RNA polymerase binds to the promoters but cannot form open complexes because of torsional inflexibility of the loop. We have shown that loop formation by GalR requires another factor, which we have purified. The purified protein behaves like the histone-like protein, called HU, of E. coli. In the absence of DNA looping, GalR bound to the upstream operator, OE, acts as an activator of P2 and a repressor of P1. GalR performs this dual role by making contacts with specific amino acid segment of the C-terminal domain of the alpha subunit of RNA polymerase bound to P2 and P1. Mutations in this region of alpha deranges the activator and/or repressor role of GalR. We have purified the GalS protein to homogeneity and studied its property. Similar to observations made in vivo, we have found that GalS mediated repression is strongest in the mgl operon, weaker in the gal operon and nearly undetectable in the galP operon. This differential behavior originates in the correspondingly different affinities of GalS toward each relevant operator DNA.